In current medical practice, the detection and analysis of biological cells have become essential in the diagnosis and treatment of many ailments. However, since a typical animal cell has a diameter of from about 10 to 20 microns and is colorless and translucent, analyzing these cells is very difficult. Most untreated cells are about 70% by weight water and are almost invisible in an ordinary light microscope, so that a current method of making these cells visible is to stain them with selected organic dyes.
The use of fluorescent markers or probes to examine cells has become very popular. Fluorescent probes have many advantages over traditional staining dyes. Fluorescent markers do not limit the analysis of microscopy specimens to the interference property of transmitted light and provide point sources of narrow band emission spectra as opposed to the transmission of full spectrum light through the entire thickness of a sample under bright field microscopy. Biological tissue often has a high degree of variability of optical properties, such as refractive index and absorption, and, as such, fluorescent markers often provide better three-dimensional imaging than bright field chromophores and have exceptional clarity for labeled objects near the objective.
If a given fluorophore provides sufficient quantum yield at wave lengths sufficiently distinct from the excitation wave length, a brighter image on a darker field will be produced. This improvement of signal-to-noise ratio can result in better resolution when imaging thick biological specimens and/or tissue sections. Additionally, a new technology for analyzing cell specimens, laser scanning confocal microscopy, requires fluorescent markers. Confocal laser scanning microscopy enables the high resolution optical sectioning of biological specimens. Optical sectioning avoids the structural artifacts and invasive nature of mechanical sectioning and permits the visualization of both living and fixed cells. Additionally, the shallow depth of field of from 0.1 to 0.5 microns of confocal laser scanning microscopes selectively limits the information gathered to a small section of the whole sample. This eliminates the background and scattered fluorescence produced by the rest of the specimen and improves the contrast, clarity and detection of the labelled structures.